M le Maire

Characterization of a high-affinity complex between the bacterial outer membrane protein FhuA and the phage T5 protein pb5.

Binding of bacteriophage T5 to Escherichia coli cells is mediated by specific interactions between the receptor-binding protein pb5 (67.8 kDa) and the outer membrane iron-transporter FhuA. A histidine-tagged form of pb5 was overproduced and purified. Isolated pb5 is monomeric and organized mostly as beta-sheets (51%). pb5 functionality was attested in vivo by its ability to impair infection of E. coli cells by phage T5 and Phi80, and to prevent growth of bacteria on iron-ferrichrome as unique iron source. pb5 was functional in vitro, since addition of an equimolar concentration of pb5 to purified FhuA prevented DNA release from phage T5. However, pb5 alone was not sufficient for the conversion of FhuA into an open channel. Direct interaction of pb5 with FhuA was demonstrated by isolating a pb5/FhuA complex using size-exclusion chromatography. The stoichiometry, 1 mol of pb5/1 mol of FhuA, was deduced from its molecular mass, established by analytical ultracentrifugation after determination of the amount of bound detergent. SDS-PAGE and differential scanning calorimetry experiments highlighted the great stability of the complex: (i) it was not dissociated by 2% SDS even when the temperature was raised to 70 degrees C; (ii) thermal denaturation of the complex occurred at 85 degrees C, while pb5 and FhuA were denatured at 45 degrees C and 74 degrees C, respectively. The stability of the complex renders it suitable for high-resolution structural studies, allowing future analysis of conformational changes into both FhuA and pb5 upon adsorption of the virus to its host.

Perfluoroalkylphosphocholines are poor protein-solubilizing surfactants, as tested with neutrophil plasma membranes.

We have tested the membrane-protein solubilizing properties of two perfluoroalkylphosphocholines. These compounds belong to a series of fluorinated amphiphiles which are being investigated as potential stabilizing agents for a variety of fluorocarbon-based systems. We are particularly interested in cytochrome b558 from phagocytes, the redox component of NADPH oxidase. Its heavy subunit is believed to carry binding sites for NADPH and FAD. Nevertheless, when the cytochrome is purified in the presence of classical detergents, it carries no FAD. This could be due to a delipidating, denaturing effect of these detergents (octyl glucoside, Triton, etc). The first perfluoroalkyphosphocholine, C8F17(CH2)2O-P(O2-)-O(CH2)2N+(CH3)3(F8C2PC), extracted about as much protein from neutrophil plasma membranes into a 100,000 g supernatant as octyl glucoside. The second compound, C8F17(CH2)11O-P(O2-)-O(CH2)2N+(CH3)3(F8C11PC), was less efficient. We found that flavin was still protein-bound in the crude F8C2PC extract at a FAD to heme ratio of about 1, and a good NADPH oxidase activity was obtained without addition of exogenous FAD, even after dialysis or gel filtration, whereas dialysis eliminated most of the FAD from the octyl glucoside extracts. These experiments appeared to make F8C2PC an interesting membrane-solubilizing agent. Nevertheless, no protein in the F8C2PC extract could be adsorbed on the chromatographic supports normally used for purification. After dilution of the extract and addition of 15 mM octyl glucoside, some of the proteins, such as myeloperoxidase, could be adsorbed (and eluted), but not cytochrome b558. Freeze-fracture electron microscopy showed that the F8C2PC extracts contained numerous vesicles and aggregates of small shapeless particles. Higher centrifugal forces sedimented most proteins of the 100,000 g supernatant. As a check, the effect of F8C2PC was tested on sarcoplasmic reticulum vesicles, the behavior of which with respect to the usual non-denaturating detergents has been well studied. There was little, if any, solubilization. We conclude that, although supernatants of F8C2PC extracts of neutrophil membranes are optically clear, proteins are not really solubilized. This result is in keeping with the absence of lytic effects of F8C2PC on erythrocyte membranes.

FhuA, an Escherichia coli outer membrane protein with a dual function of transporter and channel which mediates the transport of phage DNA.

FhuA (M(r) = 78,900) is an Escherichia coli outer membrane protein which transports the ferric siderophore ferrichrome and is the receptor for phage T5, phi 80 and T1 and for colicin M. FhuA was purified chromatographically in non-ionic detergent (octyl glucoside). The circular dichroism spectrum indicates that FhuA is essentially organized in beta-strands like the majority of proteins of the outer membrane of Gram-negative bacteria. The structural parameters of FhuA were assessed from size exclusion chromatography, sedimention equilibrium and velocity experiments. FhuA is monomeric in solution and functional since binding of phage T5 causes the release of the phage genome, a double-stranded DNA of 121,000 base pairs, into the surrounding medium. Planar lipid bilayer experiments showed that the FhuA transporter is converted into a high conductance channel upon binding of phage T5. FhuA was reconstituted into large unilamellar vesicles (mean diameter 125 nm). Cryo-electron microscopy and fluorescence experiments, using a DNA intercalant YO-PRO 1, showed that binding of T5 to FhuA triggers the transfer of the phage genome into the proteoliposomes without altering their morphology. Two models can account for these observations, which apply both to in vitro and in vivo DNA transport. The simplest model supposes that the naked DNA is transported through the FhuA channel. Alternatively transfer of DNA might be mediated by pb2, the protein forming the phage straight fiber. pb2 would insert either directly in the membrane or inside the FhuA channel.

High-performance liquid chromatography of the main polypeptide (MP26) of lens fiber plasma membranes solubilized with n-octyl β-D-glucopyranoside

The main polypeptide isolated from lens fiber membrane has been solubilized in octyl glucoside and studied by gel filtration in high‐performance liquid chromatography (HPLC). The combination of S20,w value obtained from analytical ultracentrifugation and Stokes radius determined by HPLC of the soluble fraction indicates that more than 90% of the protein is monomeric. The solubilization of the protein seems to be dependent upon the presence of the NH2 and COOH terminal sequences, since proteolytic degradation of MP26 which removes these terminal sequences is less soluble than the uncleaved polypeptide. Moreover, there is a higher amount of oligomer after proteolysis. Fatty acid analysis by gas chromatography shows that the insoluble membrane fraction from both cortical and nuclear fibers comprises a special class of long (C22) saturated fatty acids (behenic acid).

How to evaluate the distribution of an "invisible" amphiphile between biological membranes and water.

To evaluate the distribution of an amphiphile or its binding to membranes whose properties are affected by such binding, it is only necessary to establish to what extent the dose-response to the amphiphile depends on the membrane concentration. The measured response only needs to reflect local events. This method of evaluation does not depend on the precise shape of the dose-response curve and is particularly useful for amphiphiles devoid of properties like fluorescence or radioactivity which would allow their direct assay. In this work, we establish the validity of this approach by comparing it with direct conventional determinations. Two parameters are especially suitable for such evaluation: the perturbation of an enzymes activity, produced by many amphiphiles, and the fluorescence quenching of membrane-embedded proteins by chromophoric amphiphiles through long-range Förster transfer. We illustrate this approach in sarcoplasmic reticulum membranes containing Ca2(+)-ATPase as the main protein constituent. The equilibrium distribution of the antioxidant 4-nonylphenol was deduced from its inhibition of ATPase activity, whereas the equilibrium distribution of the calcium ionophore calcimycin (A23187) and of its brominated analog 4-bromo-A23187 were determined from their quenching of ATPase fluorescence. Apparent partition coefficients K* in the range of 10(5) (expressed as (moles of lipid/liter)-1) were obtained for these highly hydrophobic molecules.

Raman scattering, DRIFTS studies and Monte Carlo simulations of hexacarbonyl metals (0) encapsulated in faujasitic zeolites

Abstract Hexacarbonyl metals (0) M(CO) 6 (M=Cr, Mo, W) were sorbed at low and high coverage into faujasitic zeolites. The low-temperature DRIFTS v (CO) patterns of M(CO) 6 , occluded at low coverage depend markedly on the structure of the host and particularly on the size of the extraframework cations. At high coverage supplementary splittings are observed for zeolites with small cations in the v (CO) regions of the Raman and ATR-FTIR spectra. Monte Carlo simulations were used for predicting the sites and energetics of sorbed and cosorbed molecules at low and high loading taking into accounts the zeolite-sorbate and sorbate-sorbate interactions.

Fragmentation of dinucleotides d(ApAp) following K shell ionization of phosphorus atom by monochromatic soft X-rays

Abstract A dinucleotide: d(ApAp) with molecular formula C 70 H 61 N 11 O 15 P 2 Cl 2 has been irradiated with monochromatic soft X-rays tuned to the K shell resonance energy of the phosphorus atom. The photofragments have been analysed with an electrospray mass spectrometer. The results suggest that among all the possible electronic relaxation mechanisms, a significant one involves the scission of the two phosphodiester bonds as a result of the multiply charged phosphorus ion produced in the electronic rearrangement following the inner shell excitation or ionization of the phosphorus atom.

Vibrational spectroscopic investigations of the cation exchange and thermal activation of the silica-rich hexagonal polytype of faujasitic zeolites

Summary Infrared absorption and Raman scattering spectra were recorded after the cation exchange (K+, Cs+) of the as-synthesized silica-rich hexagonal (EMT) polytype of the faujasite zeolite. The spectra provide evidence for the conformation and the complexation of the occluded crown ether 18C6 used as template. Particularly, two types of 18C6 molecules have been detected in the dehydrated samples, corresponding probably to the two types of large cages where the molecules are occluded. The in situ vibrational spectra recorded during the thermal treatment of the as-synthesized material and the cracking pattern of 18C6 are in good agreement with ethylenic intermediates. Others applications of infrared and Raman spectroscopies in the mid and low-frequency regions to dynamics of bare EMT framework and extraframework cations of exchanged samples were evaluated as well and compared with the corresponding results concerning the silica-rich cubic faujasite, FAU, obtained using 15C5 as template.

Lipid-Protein Interactions: A Reinvestigation of Melittin Induced Effects on the Structure and Dynamics of Phosphatidylcholines

Perturbations induced by melittin (Mel) on the thermotropism of synthetic lecithins, respectively,dimyristoyl-, dipal-mitoyl- and distearoyl-phosphatidylcholine (DMPC, DPPC, DSPC) are investigated and rationalized from data obtained by fluorescence polarization (P), differential scanning calorimetry (DSC), Raman spectroscopy, and freeze-fracture electron microscopy.

Ise EF−1α associated with tubulin in X.laevis oocytes

In Xenopus laevis oocytes EF−1α is concentrated in a small region of the cytoplasm, known as the mitochondrial mass or Balbiany body. When the mitochondrial mass disperses in early vitellogenic oocytes EF−1α becomes evenely distributed in the cytoplasm. The local concentration of EF‐1α in oocytes probably results from the association of EF‐1α and EF‐1β with tubulin. Three observations are in favor of this interpretation. First, a large fraction of EF−1α in X.laevis oocytes occurs as a complex with EF−1βγ. Second, purified EF‐1γ has a strong affinity for tubulin. Third, tubulin is extremely concentrated in the mitochondrial mass and disperses exactly at the same stage of oogenesis as EF−1α. A similar association has been recently postulated between tubulin and an EF‐1α‐like protein located in the centrosphere of sea urchin eggs.